PEMT gene polymorphism and human choline requirement

This dissertation reports the identification of functional single nucleotide polymorphisms (SNPs) in the PEMT gene and their impact on choline metabolism and human dietary choline requirement. Other than dietary intake, choline can be synthesized de novo by phosphatidylethanolamine-N-methyltransferase (PEMT). PEMT knockout mice develop fatty liver and liver cell damage despite adequate dietary intake of choline. The human PEMT gene is highly polymorphic, and we speculate that functionally important polymorphisms will reduce the enzyme activity and that subjects with such SNPs will be more vulnerable to choline deficiency. In the first segment of this dissertation, we report that a common SNP in the PEMT gene, which leads to a V175M substitution, is associated with diminished activity and may confer susceptibility to nonalcoholic fatty liver disease (NAFLD). In order to find other functional SNPs, we examined the carboxyl terminus of PEMT and found that the hydrophilic C-terminus, especially the Arg at residue 189 was critical for PEMT activity. We also compared the difference in PEMT activity among four commonly used inbred mouse strains. We found that DBA2 mice had lower PEMT activity and PtdCho levels, but higher triglyceride levels in their livers. Both studies gave credence to the hypothesis that SNPs may affect PEMT function. In the last part of the dissertation we investigated whether and how the PEMT gene is regulated by estrogen and determined that the PEMT promoter has estrogen responsive elements (EREs). We found that PEMT gene transcription, protein expression and enzyme activity increased in a dose-response manner when cultured primary mouse hepatocytes were treated with various concentrations of estrogen. Estrogen significantly increased human PEMT promoter activity, and the effect was reversed by ICI 182,780, an estrogen antagonist. Eight putative EREs were found to bind with estrogen and estrogen receptor complex. A promoter SNP (rs12325817) is prevalent among women who develop fatty liver when fed a choline deficient diet. Electrophoretic mobility shift assays (EMSA) showed that this SNP reduced the binding affinity with nuclear proteins from estrogen treated cells. This study is the first to explore the underlying mechanism of why premenopausal women are resistant to choline deficiency.